Use of a compound antagonist of ESM-1 protein for producing a medicine for treating cancer

ABSTRACT

Use of an antagonist compound of protein ESM-1 for the production of a drug for the treatment of a cancer.

SCOPE OF THE INVENTION

The present invention relates to the fields of prevention and/ortreatment of cancers.

STATE OF THE ART

Despite huge financial and human investments, cancer remains one of themajor causes of death.

Cancer is frequently a disease associated with defects in the system ofintracellular signaling. Normal cells respond to numerous extracellularsignals by proliferating, differentiating or more generally changingtheir metabolic activity. Such signals are received on the surface ofthe cells and converted by a system of signal transduction proteins intoa message recognized by the cell. This message is responsible forsubsequent cell regulation phenomena.

Metastasis is the formation of a secondary tumour colony at a sitedistant from the initial tumour. It represents a multi-step process forwhich the tumoral invasion is an early event. The tumour cells escapelocally across the barrier tissues, such as the basal membrane of theepithelium, and reach the interstitial stroma, from which they gainaccess to the blood vessels or the lymph canals before subsequentdissemination. After having invaded the endothelial layer of thevascular wall, the circulating tumour cells are carried around by theblood circulation and are stopped in the precapillary venules of thetarget organ by adhesion to the lumen surfaces of the endothelial cell,or are exposed to the basal membranes. The tumour cells leave thevascular wall and enter into the parenchyma of the organ. Finally, thetumour cell, after extravasation, multiplies in a different tissue fromthat in which it originated.

It has been shown that some cancers are caused by defects associatedwith genes responsible for the transduction of the signal. Such genesare called oncogenes. These oncogenes may lead to an overexpression ofone or more signal transduction proteins inducing an abnormal cellproliferation. The defective signals may be linked to variousmechanisms.

Some anticancer therapies aim to inhibit the expression or thebioavailability of the oncogenic proteins responsible for theproliferation of the cancer cells, such as the proteins of the MAPkinase family or the products of certain oncogenes such as c-myc.

Protein ESM-1 is a polypeptide of 184 amino acids secreted by theendothelial cells and which was described for the first time by LASSALLEet al. (1996). The messenger RNAs coding for protein ESM-1 are mainlyfound in the endothelial cells and in pulmonary and renal tissues. Theexpression of the gene coding for ESM-1 is regulated by the cytokines.TNF-α and L′IL-1β induce an increase in the expression of the ESM-1 genein the endothelial cells of the human umbilical vein, while γ-Interferonreduces its expression.

A high level of circulating protein ESM-1 has been found in patientspresenting a systemic inflammatory syndrome, such as septic shock(BECHARD et al., 2000).

Current hospital treatment for cancer predominantly makes use ofradiation and/or chemotherapeutic agents, such as vinblastine oradriamycine. However, the widely known undesirable effects of suchtreatments render these strategies very difficult for the patient tosupport.

The object of the present invention is to supply anti-cancer compoundswhich overcome the disadvantages of the methods of therapeutic treatmentof cancer in the state of the art.

SUMMARY OF THE INVENTION

A first object of the invention consists of the use of an antagonistcompound of protein ESM-1 for the production of a drug for the treatmentof a cancer.

According to a first embodiment, an antagonist compound of the inventionis an antibody specifically binding to protein ESM-1.

According to a second embodiment, an antagonist compound used in thescope of the invention is a peptide of at least 10 amino acids of amodified protein ESM-1 and which contains the amino acid groupingAla(134)-Ala(135).

According to a third embodiment, an antagonist compound of protein ESM-1consists of an antisense oligonucleotide hybridizing with the cDNAcoding for ESM-1.

A further object of the invention consists of an antagonist compound ofprotein ESM-1, chosen from among the antagonist compounds defined above.

The invention also relates to a pharmaceutical composition intended forthe treatment of cancer comprising an antagonist compound of proteinESM-1.

Another object of the invention consists of a method for preventingcancer comprising a step in which an antagonist compound of proteinESM-1 is administered.

The invention also concerns a method for the therapeutic treatment ofcancer comprising a step in which an antagonist compound of proteinESM-1 is administered.

DETAILED DESCRIPTION OF THE INVENTION

It has been shown for the first time according to the invention thatprotein ESM-1 is secreted in humans in the form of a proteoglycan of thechondroitin/dermatan sulfate type and that the secreted protein ESM-1 isable to stimulate in vitro the mitogenic activity of the factor HGF/SF(Hepatocyte growth factor/scatter factor).

HGF/SF is an important factor in the appearance of renal multicysticdysplasias and in the appearance of hyperproliferation of the renaltubules and has also been associated with the development of carcinomasof the breast, kidneys and lungs and also the development of malignantmelanomas.

It has also been shown according to the invention that transfected humanrenal epithelial cells expressing protein ESM-1 have a strong tumoralpotential and cause the appearance of a renal carcinoma in vivo in mice.It has also been shown that antibodies directed against protein ESM-1were able to inhibit the development of a renal tumour in vivo and thata peptide antagonist of protein ESM-1 had the same anti-tumoralactivity.

In addition, an increase of the serum level of protein ESM-1 in patientswith a broncho-pulmonary carcinoma has been shown according to theinvention

In consequence, a first object of the invention consists of the use ofan antagonist compound of protein ESM-1 for the production of a drug forthe prevention and/or treatment of cancer.

GENERAL DEFINITIONS

The expressions “protein ESM-1” or “polypeptide of ESM-1”, in thecontext of the invention, include a polypeptide of 184 amino acidsreferenced as sequence SEQ ID N^(o)1 in the list of sequences, and alsoa polypeptide of 165 amino acids identical to the polypeptide ofsequence SEQ ID N^(o)1 in which the 19 amino acids of the N-terminal endcorresponding to the signal peptides are absent, this polypeptide of 165amino acids comprising the secreted form of the polypeptide of sequenceSEQ ID N^(o)1. Also included in the definition of “protein ESM-1” and“polypeptide of ESM-1” respectively are a glycopeptide of 184 aminoacids of sequence SEQ ID N^(o)1 and a polypeptide of 165 amino acidscorresponding to the sequence running from the amino acid in position 20to the amino acid in position 184 of the sequence SEQ ID N^(o)1 whoseserine residue in position 137 has been modified by O-glycosylation, theO-glycosylated forms of the protein ESM-1 being also designated“glycopeptides” in the present description. The ESM-1 glycopeptidepreferably has the serine residue in position 137 which isO-glycosylated by a chondroitin/dermatan sulfate group.

By “antagonist compound” of protein ESM-1, should be understoodaccording to the invention a compound able significantly to reduce thebioavailability of protein ESM-1 compared to target molecules onto whichprotein ESM-1 naturally fixes. An antagonist compound of protein ESM-1may reduce the bioavailability of these proteins by reducing theprobability of the binding of protein ESM-1 to the target molecules ofthe organism onto which it naturally fixes. An antagonist compoundaccording to the invention may reduce the bioavailability of proteinESM-1 by inhibiting or blocking the transcription of the gene coding forESM-1, by inhibiting or blocking the translation of the correspondingmessenger RNA, by modifying the intracellular maturation of proteinESM-1, for example by affecting the enzymatic process leading to itsglycosylation, or by inhibiting or blocking the secretion of the matureprotein ESM-1.

A first object of the invention consists of the use of an antagonistcompound of protein ESM-1 for the production of a drug for the treatmentof a cancer.

An antagonist compound of protein ESM-1 may be of any type, polypeptide,saccharide, or any organic or inorganic compound causing the reductionof the bioavailability of protein ESM-1 compared to the target moleculesonto which this protein fixes.

Antagonist Compounds of Protein ESM-1 of the Antibody Type

A first family of preferred antagonist compounds of ESM-1 according tothe invention is composed of antibodies specifically binding to proteinESM-1.

It has been shown according to the invention that antibodies directedspecifically against protein ESM-1 are able to inhibit or block thetumorigenic power of this protein. Anti-ESM-1 antibodies thus constituteantagonist compounds of major therapeutic value.

By “antibody” in the context of the invention, should be understood inparticular polyclonal or monoclonal antibodies or their fragments (forexample the fragments Fab or F(ab)′₂) or any polypeptide containing adomain of the initial antibody recognizing protein ESM-1.

Monoclonal antibodies may be prepared from a hybridoma according to thetechnique described by KOHLER and MIELSTEIN (1975).

They may also be antibodies directed against ESM-1 or a fragment of thisprotein produced by the trioma technique or the hybridoma techniquedescribed by KOZBOR et al. (1983).

They may also be single chain Fv antibody fragments (ScFv) such as thosedisclosed in the U.S. Pat. No. 4,9476,778 or by MARTINEAU et al. (1998).

Anti-ESM-1 antibodies according to the invention also comprise fragmentsof antibodies obtained using phage banks such as described by RIDDER etal. (1995) or human antibodies such as described by REINMANN et al.(1997) or by LEGER O J, et al., 1997.

They may also be anti-ESM-1 antibodies produced according to thetechniques described by BECHARD et al. (2000). The antibodies describedby BECHARD et al. (2000) are monoclonal antibodies secreted by hybrdomalines prepared from mouse spleen cells previously immunized against theC-terminal fragment of molecular weight 14 kD of ESM-1 which has beenproduced in Escherichia coli, in other words a nonglycosylated fragmentof protein ESM-1. By epitope mapping, BECHARD et al. (2000) were able toclassify the monoclonal antibodies produced by different hybridoma linesaccording to the region of protein ESM-1 recognized by them.

A first preferred family of antibodies according to the invention whichcomprises antagonist compounds of protein ESM-1 are the monoclonalantibodies specifically recognizing the region running from the prolineresidue in position 79 up to the cysteine residue in position 99 ofsequence SEQ ID N^(o)1, this region representing the antigenicdeterminant D1. They are preferably monoclonal antibodies produced bythe hybridoma line deposited at the Collection Nationale de Cultures deMicroorganismes of the Institut Pasteur (CNCM) under the access numberN^(o)I-1944, also named antibody MEP21.

Other preferred monoclonal antibodies are those specifically binding tothe part of protein ESM-1 contained between the glycine residue inposition 159 and the arginine residue in position 184 of sequence SEQ IDN^(o)1 which is the region comprising the antigenic determinant D3.Specific preferred monoclonal antibodies of the antigenic determinant D3may be obtained from the hybridoma line I-1943 (MEP19), deposited on 19Nov. 1997 at the Collection Nationale de Cultures des Micro-organismesof the Institut Pasteur (CNCM).

Other preferred monoclonal antibodies according to the invention are themonoclonal antibodies specifically binding to the region containedbetween the serine residue in position 119 and the valine residue inposition 139 of protein ESM-1 of sequence SEQ ID NO:1, this region beingdefined as the antigenic determinant D2 of protein ESM-1. Preferredmonoclonal antibodies specifically binding to antigenic determinant D2of ESM-1 may be obtained from the hybridoma line MEP08 deposited on 19Nov. 1997 at the Collection Nationale de Cultures de Micro-organismes ofthe Institut Pasteur (CNCM), at 28 Rue du Docteur Roux, F-75724,Paris,Cedex 15 under Accession No. I-1941.

Other monoclonal antibodies of interest constituting antagonistcompounds of protein ESM-1, within the scope of the invention, are themonoclonal antibodies specifically directed against the N-terminal partof protein ESM-1. The preferred monoclonal antibodies directed againstthe N-terminal part of protein ESM-1 may be obtained from the hybridomaline MEC15 deposited at the Collection Nationale de Cultures desMicro-organismes of the INSTITUT PASTEUR (CNCM) on 17 Oct. 2000 underthe access number I-2572.

According to a preferred embodiment, the anti-ESM1 antibodies having thebest antagonist activities against ESM-1 are chosen from among theantibodies specifically recognizing the epitopes localized in the regionaround the phenylalanine residue in position 115. They are in particularthe antibodies specifically binding to the region contained between theserine residue in position 119 and the valine residue in position 139 ofprotein ESM-1 of sequence SEQ ID N^(o)1, such as the monoclonal antibodyMEP08 described above.

It has been shown according to the invention that the monoclonalantibody MEP08 is able to inhibit the pro-tumoral activity of proteinESM-1 on the formation of tumours caused by the proliferation of humancells of renal origin in mice.

Polypeptide Antagonists of Protein ESM-1

It has been shown according to the invention that the region containingthe antigenic determinant D2 of protein ESM-1 is important for thepro-tumoral activity of protein ESM-1.

In particular, the applicant has synthesized a polypeptide derived fromprotein ESM-1 in which the phenylalanine residues in positions 134 and135 of sequence SEQ ID N^(o)1, in other words the residues in positions115 and 116 of the secreted protein ESM-1, have been replaced by twoalanine residues. The applicant has shown that this modified polypeptidewas not able to induce tumours in mice. Such a modified polypeptidecould thus compete with protein ESM-1, produced at a high level incancer patients, for its potentializing action with growth factors suchas HGF/SF or growth factors FGF-2 and FGF-7.

The antagonist compounds of protein ESM-1 include polypeptides with alength of at least 10 consecutive amino acids of sequence SEQ ID N^(o)1,which includes a sequence of amino acids running from the amino acid inposition 119 up to the amino acid in position 139 of sequence SEQ IDN^(o)1, such an antagonist polypeptide of ESM-1 containing at least onesubstitution of an amino acid, compared to the sequence corresponding toprotein ESM-1.

An antagonist polypeptide of protein ESM-1 such as defined abovepreferably has at the most 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45 or 50 consecutive amino acids of sequenceSEQ ID N^(o)1 and at least one substitution of amino acids, compared tosequence SEQ ID n^(o)1.

An antagonist polypeptide of protein ESM-1, such as defined above,contains at the most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions of anamino acid, compared to the sequence SEQ ID N^(o)1, the number ofsubstitutions of amino acids being adapted as a function of the lengthof the polypeptide, it being understood that the number of substitutionsof amino acids compared to the sequence SEQ ID N^(o)1 in an antagonistpolypeptide according to the invention is at the most 25% of the aminoacids contained in the sequence of this antagonist polypeptide,preferably at most 20%, 15% and more preferably at most 10% of thenumber of amino acids contained in the sequence of the antagonistpolypeptide of ESM-1.

A substitution of amino acids, compared to the sequence SEQ ID N^(o)1,in an antagonist polypeptide according to the invention is s preferablya “non-conservative” substitution. By “non-conservative” substitutionshould be understood the substitution of an amino acid residue by anamino acid of a different class.

Amino acids are conventionally classified according to the followingclasses:

-   -   non polar amino acids (hydrophobic): alanine, leucine,        isoleucine, valine, proline, phenylalanine, tryptophan and        methionine;    -   amino acids containing aromatic rings: phenylalanine, tryptophan        and tyrosine;    -   neutral polar amino acids: glycine, serine, threonine, cysteine,        tyrosine, asparagine and glutamine;    -   positively charged amino acids (basic): arginine, lysine and        histidine);    -   negatively charged amino acids (acid): aspartic acid and        glutamic acid.

A preferred type of substitution of amino acids for the preparation ofan antagonist polypeptide of protein ESM-1 according to the invention isthe substitution of an amino acid containing an aromatic ring by anamino acid not containing an aromatic ring.

An antagonist polypeptide of protein ESM-1 according to the inventionpreferably contains a substitution of the phenylalanine residues inpositions 134 and 135 of SEQ ID N^(o)1 by two amino acid residues,identical or different, not containing an aromatic ring.

Such a preferred antagonist polypeptide of protein ESM-1 is apolypeptide of at least 10 consecutive amino acids of sequence SEQ IDN^(o)1, such as defined above, in which the phenylalanine residues inpositions 134 and 135 have been replaced by two alanine residues.

According to a first embodiment, an antagonist polypeptide of proteinESM-1 according to the invention may be prepared by conventionalchemical synthesis techniques, either in homogenous solution or in thesolid phase.

As an illustration, an antagonist polypeptide of protein ESM-1 may beprepared by the homogeneous solution technique described by HOUBEN WEIL(1974) or by the solid phase synthesis technique described by MERRIFIELD(1965a; 1965b) and MERRIFIELD 1965b.

An antagonist polypeptide of protein ESM-1 according to the inventionmay also be prepared by genetic recombination.

In order to produce an antagonist polypeptide of protein ESM-1 such asdefined above, a method may be used comprising the steps of:

a) inserting a nucleic acid coding for the antagonist polypeptide ofprotein ESM-1 in an appropriate expression vector;

b) culturing, in an appropriate culture medium, a host cell previouslytransformed or transfected with the recombinant expression vector ofstep a);

c) recovering the culture medium or lysing the host cell, for example bysonication or osmotic shock;

d) separating and purifying from said culture medium or cell lysatesobtained in step c), said antagonist polypeptide;

e) if appropriate, characterizing the recombinant antagonist polypeptidethus produced.

The antagonist polypeptides according to the invention may becharacterized by fixation on an immunoaffinity chromatography column onwhich the antibodies directed against this polypeptide or against afragment of it have previously been immobilized.

According to another embodiment, an antagonist polypeptide of ESM-1 maybe purified by passage over an appropriate series of chromatographycolumns, according to methods known to a person skilled in the art anddescribed for example by AUSUBEL F. et al. (1989).

Antagonist Compounds of Protein ESM-1 of the Antisense OligonucleotideType.

Another preferred family of antagonist compounds of protein ESM-1 aimingto reduce the bioavailability of protein ESM-1 secreted in patients atrisk or in patients having already developed tumours are compounds ableto inhibit or block the expression of the gene coding for ESM-1 inhumans.

Such antagonist compounds of protein ESM-1 may be antisensepolynucleotides.

The antagonist compounds of protein ESM-1 according to the inventionthus include an antisense polynucleotide able to hybridize specificallyto a given region of the gene coding for protein ESM-1 and able toinhibit or to block its transcription and/or its translation.

The sequence of the human ESM-1 gene is referenced under the accessnumber AJ401 1091 and AJ401 1092 in the database Genbank.

An antisense polynucleotide according to the invention preferablycontains a sequence complementary to a sequence localized in the regionof the 5′-end of the DNA of the ESM-1 gene, and more preferably close tothe initiation codon of the translation (ATG) of the ESM-1 gene.

According to a second preferred embodiment, an antisense polynucleotideaccording to the invention contains a sequence complementary to one ofthe sequences localized at the exon/intron junctions of the ESM-1 geneand preferably sequences corresponding to a splicing site.

A preferred antisense polynucleotide according to the invention containsat least 15 consecutive nucleotides of the cDNA coding for ESM-1 havingthe nucleotide sequence SEQ ID N^(o)2.

For the purposes of the present invention, a first polynucleotide isconsidered as being “complementary” to a second polynucleotide when eachbase of the first nucleotide is paired with the complementary base ofthe second polynucleotide whose direction is inversed. The complementarybases are A and T (or A and U), and C and G.

In general, an antisense polynucleotide according to the invention hasat least 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000or 2000 consecutive nucleotides of the cDNA of ESM-1 of sequence SEQ IDN^(o)2.

As an illustration, a preferred antisense polynucleotide according tothe invention consists of a nucleic acid of complementary sequence tothe nucleic acid of the cDNA of ESM-1 of sequence SEQ ID N^(o)2.

An antisense polynucleotide comprising an antagonist compound of proteinESM-1 according to the invention may be prepared by any suitable methodwell known to a person skilled in the art, including cloning and theaction of a restriction enzyme or by chemical synthesis according totechniques such as the phosphodiester method of NARANG et al. (1979) orof BROWN et al. (1979), the diethylphosphoramidite method of BEAUCAGE etal. (1980) or the solid support technique disclosed in the Europeanpatent n^(o)EP-0 707 592.

In general, antisense polynucleotides must have a length and a meltingpoint sufficient to allow the formation of an intracellular duplexhybrid having sufficient stability to inhibit the expression of the mRNAof ESM-1. Strategies to construct antisense polynucleotides are inparticular described by GREEN et al. (1986) and IZANT and WEINTRAUB(1984).

Methods for construction of antisense polynucleotides are also describedby ROSSI and al (1991) and in the PCT applications N^(o)WO 947/23.026,WO 95/04141, WO 92/L18.522 and in the European patent application n^(o)EP 0 572 287.

Other methods for the use of antisense polynucleotides are for examplethose described by SCZAKIEL et al. (1995) or those disclosed in the PCTapplication N^(o)WO 95/24,223.

A skilled person may advantageously refer to the methods of productionand use of antisense polynucleotides inhibiting or blocking theexpression of genes associated with the development of cancers, such asthe techniques disclosed in the U.S. Pat. No. 5,582,986 which disclosesantisense oligonucleotides for inhibiting the ras gene, the techniquedescribed by HOLT et al. (1988) which describes antisenseoligonucleotides specifically hybridizing with messenger RNAs of theoncogene c-myb or the technique described by WICKSTRON et al. (1988)which describes antisense oligonucleotides specifically hybridizing withthe messenger RNA of the gene c-myc.

Other techniques for using antisense polynucleotides usable by a skilledperson are those of SALE et al. (1995) and that of GAO et al. (1996).

Method for Selecting an Antagonist Compound of Protein ESM-1

An antagonist compound of protein ESM-1 according to the invention maybe selected by a person skilled in the art for its capacity to inhibitthe development of a tumour induced by protein ESM-1 in vivo.

According to a first embodiment, a method for selecting an antagonistcompound of protein ESM-1 comprises the following steps:

a) injecting an animal with cells able to form tumours in the presenceof protein ESM-1, said cells being transfected or transformed by anucleic acid able to express protein ESM-1 in vivo;

b) administering to this animal a candidate antagonist compound ofprotein ESM-1;

c) comparing the formation of tumours in a first animal such as obtainedafter step b) and in a second animal such as obtained after step a); and

d) selecting the candidate compound able to inhibit or block theformation of tumours in the first animal.

The animal used in the selection method above is preferably a non-humanmammal, advantageously a rodent, and more preferably a rat, guinea pigor mouse.

In a particular embodiment of the method, this includes a step e)consisting of sacrificing the first and the second animal.

Advantageously, the cell line able to form tumours in the animal in thepresence of protein ESM-1 is the line HEK 293 (ATCC N^(o)CRL 1573).

According to a further embodiment, an antagonist compound of proteinESM-1 according to the invention may be selected according to a methodusing the demonstration of the fixation of a candidate compound ontoprotein ESM-1. Such a method of selection of a candidate antagonistcompound of protein ESM-1 comprises the following steps:

a) supplying a polypeptide consisting of protein ESM-1 or a peptidefragment of this protein;

b) placing said polypeptide in contact with the candidate compound to betested;

c) detecting the complexes formed between said polypeptide and thecandidate compound;

d) selecting the candidate compounds fixing onto the polypeptideconsisting of protein ESM-1 or a peptide fragment of this protein.

By “fragment” of protein ESM-1, should be understood a polypeptidecontaining at least 20, preferably at least 30, 35, 40, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140 or 150 consecutive aminoacids of the polypeptide ESM-1 of sequence SEQ ID N^(o)1 and containingthe sequence running from the proline residue in position 133 up to thevaline residue in position 138 of SEQ ID N^(o)1.

The invention also relates to a kit for selecting a candidate antagonistcompound of protein ESM-1, this kit comprising:

a) a purified preparation of a polypeptide consisting of protein ESM-1or of a fragment of this protein;

b) where appropriate, means of detection of a complex formed between thepolypeptide and the candidate compound to be tested.

The method of detection of a complex formed between the polypeptidederived from protein ESM-1 and the candidate compound may be performedby various techniques, such as microdialysis coupled with an HPLC methodas described by WANG et al. (1997) or affinity capillary electrophoresisas described by BOUSH et al. (1997).

A candidate compound may be of any type, and particularly the finalproduct of a combinatorial chemistry method.

A. Candidate Compounds Obtained from Peptides Banks

A candidate antagonist compound of protein ESM-1 may be selectedaccording to the method above as an expression product of a DNA insertcontained in a phage vector according to the technique described byPARMLEY & SMITH (1988). In this type of peptide bank, the DNA insertscode for peptides of 8 to 20 amino acids in length, as is described byOLDENBURG K R et al. (1992), VALADON P et al. (1996), LUCAS A H (1994),WESTERINK (1995), FELICI et al. (1991).

According to this particular embodiment, the recombinant phagesexpressing a protein able to fix onto the polypeptide consisting ofprotein ESM-1 or a fragment of it are retained and the complex formedbetween protein ESM-1 or a fragment of it and the recombinant phage maybe subsequently immunoprecipitated by an anti-ESM-1 monoclonal orpolyclonal antibody.

B. Candidate Compound Obtained by Competition Experiments

The candidate antagonist compounds of protein ESM-1 may also be selectedby the fact that they fix onto protein ESM-1, or onto a polypeptidefragment of it, in competition with a previously selected antagonistcompound of protein ESM-1 such as one of the anti-ESM-1 antibodiesdescribed above, and particularly the monoclonal antibody secreted bythe hybridoma line MEPOB deposited on 19 Nov. 1997 at the CNCM under theaccess number 1-1941.

Such competition experiments are for example described in the article byBECHARD et al. (2000).

C. Candidate Antagonist Compounds of Protein ESM-1 Selected by AffinityChromatography.

Proteins or other molecules of any type able to fix onto protein ESM-1,or to a polypeptide fragment of this protein, may be selected by usingaffinity columns on which protein ESM-1 or a fragment of it havepreviously been immobilized, for example by conventional techniques,including the chemical coupling of protein ESM-1 or a fragment of itwith the matrix of a column such as of agarose, or AffiGel®. A solutioncontaining the candidate compound to be tested is placed in contact withthe chromatographic support on which protein ESM-1 or a peptide fragmentof it has been immobilized. The compounds retained on the affinitycolumn are positively selected.

D. Candidate Compounds Selected by Optical Biocaptor Techniques

A candidate antagonist compound of protein ESM-1 may also be selected byusing an optical biocaptor such as described by EDWARDS andLEATHERBARROW (1997). This technique allows the detection ofinteractions between molecules in real time without the necessity ofusing marked molecules. This technique is based on SPR (Surface PlasmonResonance). Briefly, the candidate compound to be tested is fixed onto asurface, such as a carboxymethyidextran matrix. A light ray is directedonto the part of the surface which does not contain the sample to betested and is reflected by this surface. The SPR phenomenon causes areduction in the intensity of the reflected light with a specificassociation between the angle of the reflected light and the wavelengthof the light ray. The fixation of the candidate compound causes a changein the refractive index of the surface, the change in the refractiveindex being detected as a modification of the SPR signal.

Such a detection method by optical biocaptor may also permit theselection of candidate compounds which enter into competition withanother ligand for the fixation onto protein ESM-1 or a peptide fragmentof it.

For example, a candidate antagonist compound of protein ESM-1 includescompounds able to inhibit the fixation of an anti-ESM-1 antibody ontoprotein ESM-1, to inhibit the fixation of factor HGF-SF or factors FGF-2and FGF-7 onto protein ESM-1 or a peptide fragment of this protein.

Thus, according to a further embodiment, the invention relates to amethod of selection of an antagonist compound of protein ESM-1characterized in that it comprises the following steps:

a) Placing protein ESM-1 or a peptide fragment of it in contact with:

(i) an antagonist compound of protein ESM-1 fixing onto protein ESM-1;and

(ii) a candidate compound to be tested;

b) in a separate step from step a), but optionally simultaneously withit, placing protein ESM-1 or a peptide fragment of it in contact with anantagonist compound of protein ESM-1 fixing onto protein ESM-1;

c) detecting the respective quantity of antagonist compound of proteinESM-1 fixed after each of steps a) and b); and

d) selecting the candidate compound which enters into competition withthe antagonist compound for the fixation onto protein ESM-1.

An antagonist compound of ESM-1 for the use of the selection methodabove is preferably an anti-ESM-1 antibody or a peptide antagonistcompound such as defined above in the present description.

In a first embodiment of a method for selecting an antagonist compoundof ESM-1 from a candidate compound, said method comprises the followingsteps:

1) selecting, among the candidate compounds, the compounds which fixonto protein ESM-1 or onto a peptide fragment of this protein;

2) administering a compound selected in step 1) to an animal anddetermining the capacity of this compound to inhibit, in this animal,the development of tumours induced by protein ESM-1;

3) selecting the compounds which inhibit the development of tumoursdetermined in step 2) as antagonist compounds of protein ESM-1.

Step 1) preferably consists of the use of a selection method of acandidate compound fixing onto protein ESM-1 or onto a peptide fragmentof this protein, chosen from among the methods detailed in the presentdescription.

Step 2) preferably consists of the use of a selection method of acandidate compound in vivo such as is detailed in the description.

In a particular embodiment of the method, this also contains a step 4)consisting of sacrificing the animal.

Pharmaceutical Composition of the Invention.

A further object of the invention is a pharmaceutical composition forthe treatment and/or prevention of a cancer containing an antagonistcompound of protein ESM-1.

Pharmaceutical Composition Containing an Antagonist Compound of theAntibody Type or of the Peptide Type According to the Invention.

According to a first embodiment, a pharmaceutical composition accordingto the invention contains a therapeutically effective quantity of ananti-ESM-1 antibody or of a peptide antagonist compound derived fromESM-1, in combination with one or more pharmaceutically compatiblevehicles. The pharmaceutical compositions according to the inventioninclude those suitable for topical, oral, rectal, nasal or parenteral(including intramuscular, subcutaneous and intravenous) administrationor in a form suitable for administration by inhalation or insufflation.The pharmaceutical compositions according to the invention may bepresented in the form of unit doses and may be prepared by any methodwell known to a person skilled in the art of pharmaceutical medicine Allthe methods include a step consisting of combining the antagonistcompound comprising the active principle of the composition with aliquid vehicle or a finely divided solid vehicle and, if necessary,forming the product, for example in the form of tablets or capsules.

For oral administration, a pharmaceutical composition according to theinvention is preferably presented in the form of dose units such astablets, capsules or hard capsules. When it is presented in a formcontained in a pressurized container, the pharmaceutical composition maycontain a propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother appropriate gases. In the case of a pressurized aerosol, the doseunit may be provided with a valve able to supply a given quantity of thepharmaceutical composition.

According to another embodiment, the pharmaceutical compositionaccording to the invention may be in the form of a dry powdercomposition for administration by inhalation or insufflation, forexample in the form of a mixture of a powder of the antagonist compoundand of a suitable base powder, such as lactose or starch. The powdercomposition may be presented in a dose unit, for example in s the formof capsules or dispensers from which the powder may be administeredusing an inhaler or insufflator device.

A solid pharmaceutically acceptable vehicle compatible with apharmaceutical composition according to the invention includessubstances such as flavouring agents, lubricants, solubilizing agents,suspension agents, fillers, compression auxiliaries, binders ordispersion agents as well as encapsulating materials. In the powders,the vehicle is a finely divided solid which is in admixture with theantagonist compound of ESM-1 also in a finely divided form. In thetablets, the active principle antagonist of ESM-1 is mixed with avehicle having suitable compression properties and compacted into thedesired form and size. The powders and tablets preferably contain lessthan 99% of the active principle. The preferred solid vehicles are forexample calcium phosphate, magnesium stearate, talc, sugars, lactose,dextrin, starch, gelatine, cellulose, polyvinylpyrrolidone and theionexchange resins.

Liquid vehicles are used to prepare a pharmaceutical compositionaccording to the invention in the form of a solution, a suspension, anemulsion, a syrup, an elixir and a pressurized composition. The activeprinciple antagonist of protein ESM-1 may be dissolved or suspended in apharmaceutically acceptable vehicle such as water, an organic solvent,or a mixture of the two or pharmaceutically acceptable oils or fats. Theliquid vehicle may contain other pharmaceutically acceptable additivessuch as solubilizing agents, emulsifiers, buffers, preservatives,sweeteners, flavouring agents, suspension agents, thickening agents,colorants, viscosity regulators, stabilizers or osmo-regulators.Illustrative examples of liquid vehicles for oral and parenteraladministration include water, alcohols, (including monohydric andpolyhydric alcohols such as the glycols), oils such as coconut oil orfractionated peanut oil. For parenteral administration, the vehicle mayalso be an ester such as ethyl oleate and isopropyl myristate. Liquidpharmaceutical compositions in the form of sterile solutions orsuspensions may be used for intramuscular, intraperitoneal orsubcutaneous injection.

A pharmaceutical composition according to the invention preferablycontains from 1 to 1000 mg of antagonist compounds of protein ESM-1 perdose unit, and preferably from 10 to 500 mg of antagonist compound ofprotein ESM-1 per dose unit.

The present invention also concerns a method of treatment and/orprevention of a cancer comprising a step during which a pharmaceuticalcomposition such as defined above is administered to a patient havingneed of such treatment.

Pharmaceutical Composition Containing an Antagonist Compound of ProteinESM-1 of the Antisense Polynucleotide Type.

Also forming part of the invention are pharmaceutical compositionscontaining a therapeutically effective quantity of an antagonistcompound of protein ESM-1 of the antisense polynucleotide type asdefined in the present description in addition to methods of treatmentand/or prevention of a cancer comprising the administration to a patienthaving need of such a treatment of a pharmaceutical compositioncontaining an antisense polynucleotide such as defined above.

An antisense oligonucleotide according to the invention may beadministered by any means, either local or systemic.

The local administration of an antisense polynucleotide of theinvention, for example in the tumour, may be performed by theadministration of the antisense polynucleotide directly into the tumouror into the tissue surrounding the tumour so that the oligonucleotidecan migrate to, and where appropriate enter into, the tumour cells. Forexample, the antisense polynucleotides may be injected using a syringe.The injection may be intramuscular, intravenous, intraperitoneal orsubcutaneous. The antisense polynucleotide may be administered to theliver via the hepatic portal vein. Similarly, the antisensepolynucleotide may be administered to the lung using an inhalationdevice.

Other means of administration of an antisense polynucleotide may beused. For example, the antisense polynucleotides may administeredsystemically after their insertion into an expression vector. The term“expression vector” includes a plasmid, a virus or any other vehicleknown in the state of the art to ensure the expression of an antisensepolynucleotide.

For the use of vectors suitable for the recombinant expression of anantisense polynucleotide, a person skilled in the art may advantageouslyuse the vectors pMSXND described by LEE and NATHANS (1988), eukaryoticvirus vectors, such as those described by GLUZMAN (1982), or theadenoviruses and adeno-associated viruses such as those described in theU.S. Pat. Nos. 5,173,414 and 5,354,678 or an expression system includingan expression vector described by MOXHAM et al. (1993).

The expression vector preferably contains a promoter allowing theproduction of the antisense polynucleotide in an animal, preferably amammal, and preferably in humans, such as the polyhedrin promoter.

The expression vector may be suitable for the targeted expression of theantisense polynucleotide at the site of the tumour, for example byplacing the nucleic acid coding for the antisense polynucleotides underthe control of a promoter specific to certain cells, such as theepithelial cells or the endothelial cells. An example of such a promoteris the viral promoter designated NuNTV which is specifically useful inthe treatment of breast cancers. Other examples of such specificpromoters are milk protein promoters such as β-lactoglobulin, α-caseinand β-casein.

The therapeutically effective quantity of an antisense polynucleotide ofthe invention may be determined as the quantity necessary for asignificant reduction of the translation of protein ESM-1 at thesystemic or local level.

It will be clear to a person skilled in the art that the therapeuticallyeffective concentration of the antisense potynucleotide varies with thechoice of the mode of administration. For example, if the antisensepolynucleotide is administered by injection to a mammal, the dose unitcomprises a syringe containing an effective quantity of the antisensepolynucleotide. An effective quantity of the antisense polynucleotidefor a systemic administration is between 0.01 mg/kg and 50 mg/kgadministered once or twice per day. A therapeutically effective quantityof an antisense polynucleotide according to the invention included in apharmaceutical composition is generally between 10⁴ and 10¹¹ moleculesof antisense polynucleotide per administration and preferably between10⁵ and 10¹⁰ molecules of DNA per administration.

However, different dosage protocols may be used according to (i) theindividual capacity of the antisense polynucleotide to inhibit theexpression of protein ESM-1, (ii) the severity or extent of the disease,or (iii) the pharmacokinetic behaviour of the antisense polynucleotideused.

The antisense polynucleotide may be combined with a pharmaceuticallyacceptable vehicle or an excipient. Examples of excipients includefillers, binders, dispersion agents, lubricants, according to the typeof administration and the forms of dosage. Preferred forms of dosageinclude liquid solutions, advantageously physiologically compatiblebuffers such as HANK's or RINGER solutions. In addition, the antisensepolynucleotides according to the invention may be formulated in a solidform then redissolved or resuspended immediately before use. Thisincludes lydphilized forms and liposomes containing such antisensepolynucleotides.

An antisense polynucleotide of the invention may also be systemicallyadministered by the transmucosal, transdermal or oral routes. For thetransmucosal or transdermal routes of administration, penetrating agentsmay be used in formulation such as bile salts or derivatives of fusidicacid.

The present invention also relates to a method of treatment and/orprevention of a cancer comprising a step of administration, to a patienthaving need of such treatment, of a pharmaceutical composition such asdefined above containing an antagonist compound of ESM-1 of theantisense polynucleotide type.

In general, any of the pharmaceutical compositions of the invention suchas defined above and containing a therapeutically effective quantity ofan antagonist compound of protein ESM-1 is useful in the preventionand/or treatment of a cancer.

As a non-limiting illustration, a pharmaceutical composition accordingto the invention is useful for the prevention and/or treatment ofcancers such as cancers of the respiratory tracts, broncho-pulmonarycancers, breast cancers, cancers of the colon and renal cancers as wellas cancers of the digestive system.

The present invention is in addition illustrated, without in any waybeing limited, by the following examples and figures.

FIGURES

FIG. 1 illustrates Western Blot immunoblotting gels and colorations ofESM-1 on SDS-PAGE gel.

Each immunoblotting gel was revealed with the anti-ESM-1 monoclonalantibody MEP14. The second anti-mouse antibody marked with horseradishperoxidase was purified by affinity and gave negative results when usedalone.

FIG. 1A. Immunoblotting gel of protein ESM-1 from different cell typesexpressing this protein.

The immuno-precipitation of ESM-1 from cell culture supernatants SVI(1), 293-ESM(2) and CHO-ESM(3) was performed with the antibody MEP19when this is indicated, or with a control antibody. The arrows show theband specific to ESM-1. The native form of ESM-1 is represented by adiffuse band around 50 kD.

FIG. 1B. Absence of detection of purified protein ESM-1 with coomassieblue.

5 μg of protein ESM-1 purified from SVI cells was loaded onto anSDS-PAGE gel at 15% and coloured with coomassie blue in order to detectthe peptide part of the molecule. The arrows show the absence ofdetection of ESM-1.

FIG. 1C. Detection of purified protein ESM-1 with alcian blue.

5 μg of protein ESM-1 purified from SVI cells was loaded onto anSDS-PAGE gel at 15% and revealed with alcian blue in order to detect theglycan part of the molecule. The arrow shows protein ESM-1.

FIG. 2 illustrates the apparent molecular weight of the peptide andglycan parts of ESM-1.

FIG. 2A Analysis by mutation of the site of fixation of O-glycosylation.

Two presumed O-glycosylation sites (threonine 120 and serine 137) weresubstituted by an alanine residue by directed mutagenesis. The wild-typeprotein ESM-1 (VT), the ESM-1 T120A and S137A mutants, and negativecontrols (MOCK) were transfected in 293 cells and the cell culturesupernatants and cell lysates were analysed by immunoblotting(Western-Blot) using monoclonal antibody MEP14. The arrows show thespecific bands,

FIG. 2B. Effect of a treatment with proteinase K on ESM-1.

Protein ESM-1 purified from SVI (1) and 293-ESM cells (2) was digestedby proteinase K and loaded onto an SDS-PAGE gel at 15% The upper arrowshows the wild type of untreated protein ESM-1 and the lower arrow showsprotein ESM-1 digested by proteinase K.

FIG. 3 illustrates the effects of specific chondroitinases on ESM-1.

FIG. 3A. Treatment of purified wild-type protein ESM-1 withchondroitinase ABC.

Secreted protein ESM-1 was purified by ion-exchange chromatography,followed by immunoaffinity chromatography from cell culture supernatantsof SVI(1), 293ESM(2) and from human plasma (3), then digested or not bychondroitinase ABC. 50 ng of the digested protein were loaded onto anSDS-PAGE gel at 15% then analysed by immunoblotting (Western-Blot). Theupper arrow shows the undigested forms of ESM-1 and the lower arrow thedigested forms of ESM-1.

FIG. 3B. Treatment of purified wild-type protein ESM-1 withchondroitinase B.

Protein ESM-1 purified from cell culture supernatants of SVI (1) and293-ESM(2) was digested or not by the chondroitinase. The proteins wereloaded onto SDS-PAGE gel at 15%. The upper arrow shows the differentundigested forms of ESM-1 around 50 kD and the lower arrow shows thedifferent forms of digested ESM-1, around 22 kD.

FIG. 3C. Treatment of purified wild-type protein ESM-1 withchondroitinase AC.

Protein ESM-1 purified from cell culture supernatants of HUVEC (1) and293-ESM(2) was digested by chondroitinase AC and loaded onto SDS-PAGEgel at 15%. The upper arrow shows the different undigested forms ofESM-1 around 502 kD and the lower arrow shows the different forms ofdigested ESM-1, around 22 kD.

FIG. 3D. Treatment of purified wild-type protein ESM-1 withchondroitinase C.

Protein ESM-1 purified from cell culture supernatants of HUVEC (1) and293-ESM (2) was digested or not by chondroitinase C and loaded ontoSDS-PAGE gel at 15%. The upper arrow shows the different undigestedforms of ESM-14 around 52 kD and the lower arrow shows the differentforms of digested ESM-1 around 22 kD.

FIG. 4 illustrates the effects of purified wild-type protein ESM-1 onthe coagulation time in the presence of thrombin. The delay and thereduction of thrombin production can be seen for heparinized plasma andalso for the four other curves (plasma rich in platelets or PRP: opendiamonds; plasma rich in platelets+ESM-1 at 0.2 mg/ml: solid squaresplasma rich in platelets+ESM-1 00.5 mg/ml: solid triangle; plasma richin platelets+ESM-1 at 1 mg/ml: solid circle; plasma rich inplatelets+heparin: open circle).

FIG. 5 illustrates the biological activity of the proteoglycan ESM-1 onthe proliferation of 293 cells induced by factor HGF/SF. The stimulationof the incorporation of ³H-thymidine by 293 cells induced by factorHGF/SF was studied. The cells were sown at 1×10⁴ cells per well in aDMEM medium supplemented with transferin and insulin and HGF/SF at 50 ngper ml before addition of different molecules. The bars represent thepercentage increase in ³H-thymidine incorporation (mean+/−s.d. of triplesamples of a representative experiment) in the presence of the additionsshown of serum, different forms of ESM-1 at 2.5 mg/ml and decorin at 2.5mg/ml. The background noise level of ³H-thymidine incorporation in thepresence of HGF/SF was generally between 7.000 and 8.000 cpm per well.The results presented are similar to those obtained in three otherseparate experiments.

FIG. 6 illustrates a study of twelve responses of the different forms ofESM-1 and of decorin on the mitogenic activity induced by factor HGF/SF.The simulation of DNA synthesis by 293 cells was studied in the presenceof HGF/SF at 50 nanograms per ml alone or in the presence of differentconcentrations of wild-type protein ESM-1/WT (open square), of mutantnon-glycosylated protein ESM/S137A (solid circle), of the GAG chainderived from protein ESM/WT (solid square) or of decorin (open circle).The mean values of triplicate measurements of ³H-thymidine incorporationobtained in one experiment among three independent experiments are shownin FIG. 6. The results are expressed in cpm. The standard deviationswere approximately 10%.

FIG. 7 illustrates the tumorigenic power of protein ESM-1. Two batchesof more than 10 mice received control HEK cells or cells transfectedwith a vector coding for the cDNA of wild-type protein ESM-1 (ESM/WT).On FIG. 7A, the percentage of tumours macroscopically visible at theeighth week at the injection point and whose tumoral volume was morethan 1 cm³ is shown as ordinate. FIG. 7B illustrates the kinetics ofappearance of the tumours in mice having received transfected HEKexpressing glycosylated protein ESM-1 (ESM/WT). The number of weeksafter injection of the cells is given on the abscissa. The mean tumoralvolume, expressed in cm³, is given on the ordinate.

FIG. 8 illustrates the production of ESM-1 by tumours induced in mice.

FIG. 8A represents the serum level of protein ESM-1 found in the twobatches of mice, at the eighth week following the injection of thecells. The abscissa shows respectively the batch of mice having receivedthe control HEK cells and the batch of mice having received the HEKcells expressing glycosylated protein ESM-1 (ESM/WT). The serum level ofESM-1 found, expressed in nanogram/ml, is given on the ordinate.

FIG. 8B illustrates the kinetics of the serum levels of ESM-1 measuredby ELISA, for the mice of the batch having received the HEK cellstransfected with a DNA coding for the glycosylated protein ESM-1(ESM/WT). The abscissa shows the number of weeks following the injectionof the transfected cells The serum level of protein ESM-1, expressed innanogram/ml, is given on the ordinate.

FIG. 9 illustrates the tumorigenic activity of the different forms ofprotein ESM-1.

FIG. 9A illustrates the appearance of tumours in different batches ofmice, the mice having received respectively control HEK cells, HEK cellstransfected with a cDNA coding for glycosylated protein ESM-1 (ESM/WT),cells transfected with nonglycosylated protein ESM-1 (ESM/S137A) and HEKcells transfected with a cDNA coding for protein ESM-1 replaced inpositions 134 and 135 (ESM/F115A, F116A). The ordinate shows thepercentage of tumours macroscopically visible at the eighth week at thepoint of injection whose tumoral volume was greater than 1 cm³.

FIG. 9B illustrates the serum level of ESM-1 in the different identicalbatches of mice. The serum level of ESM-1, expressed in nanogram/ml, isgiven on the ordinate.

FIG. 10 illustrates the inhibiting effect of the monoclonal antibodyMEP08 on the pro-tumoral activity of protein ESM-1.

The injection of MEP-08 antibodies increased the survival of mice fromthe HEK ESM/WT group. The monoclonal antibodies MEP-08 were injectedintraperitoneally at a dose of 400 μg from the second following theinoculation of the HEK/ESM-WT cells. The injections were repeated weeklyfor 12 weeks. A control antibody, MEP-14, was used under the sameconditions. The mice were sacrificed when their tumoral volume wasgreater than 6 cm³. (n>8 mice in each group). The figure shows thepercentage of surviving mice in each of the groups.

EXAMPLES Example 1 Post-translational Modification of the Secreted Formof Protein ESM-1

A. Materials and Methods

A.1 Cell Culture and Materials

CHO cells were cultured in a culture medium MAMα (Gibco BRL, LifeTechnologies, France) supplemented with 10% foetal calf serum. Humanendothelial cells transfected by the virus SV40, the SVI cells describedby LASSALLE P et al. (1992), were cultured in a medium RPMI 1640containing 2 mM of L-glutamine and 10% foetal calf serum. Human embryokidney cells, the cells of the line 293, were cultured in a medium DMEMfrom Dulbecco with 10% foetal calf serum. The human embryo kidney cells,the cells of the line 293, used for the proliferation test were culturedin modified EAGLE medium from Dulbecco (Gibco BRL) supplemented withinsulin at 10 mg/ml and transferin at 10 mg/ml. The proteinase andchondroitinase ABC were commercially available from Boehringer Mannheim.Chondroitinases B, AC and C are marketed by Sigma. Human factor HGF/SFis marketed by R & D and decorin by Sigma. Anti-ESM-1 monoclonalantibodies were produced and purified as described by BECHARD et al.(2000).

A.2 Development of Cell Lines Expressing ESM-1.

The complete cDNA coding for ESM-1 was directed, purified and insertedinto the expression vector pcDNA3 (marketed by Invitrogen) between theXhoI and HindlIII sites. The vector constructions were transfected inthe cell lines CHO and 293 in the presence of lipofectamine (GIBCO BRL),then selected on G418 (1000 μg/ml for the CHO line and 300 μg/ml for the293 line). The cell lines which had been transfected stably wereobtained by limiting dilution and the cells thus selected weredesignated respectively CHO-ESM and 2936-ESM.

A.3 Determination of the Site of O-lycosylation of ESM-1 by MutationAnalysis.

2 Potential sites of Olycosylation had been predicted using the softwareNET 0 glyc:0 Prediction Serveur.

The serine residue in position 137 (SEQ ID N^(o)1) and the threonine inposition 120 (SEQ ID N^(o)1) were substituted by an alanine residue. TheO-glycosylation mutants were produced by PCR using the mutagenesis kitQuick Change according to the manufacturer's recommendations(Stratagene).

The mutant cDNAs were confirmed by sequencing (sequencer ABI prism 377from Applied Biosystems). The 293 cells were then transfected with thevectors into which the mutant cDNAs had been inserted to obtain thetransitory and stable transfectants, respectively the 293-ESM/S 137A and293-ESM/T120A.

A.4 Purification of the Proteoglycan ESM-1 Chondroitin/dermatan Sulfate.

The cell culture supernatants were adjusted to pH8, then passed over acolumn of DEAE-Sepharose (Pharmacia), washed with a Tris buffer 50 mM(pH8), 0.2 M NaCl, then eluted with a buffer Tris 50 mM (pH8), 0.8 MNaCl.

The eluates were adjusted to 50 mM Tris (pH8), 0.5 M NaCl and passedover an affinity column. The affinity column was composed of anti-ESM-1monoclonal antibodies (produced by the hybridoma line MEC4) immobilizedon a Affigel Hz hydrazide gel, according to the manufacturer'srecommendations (Biorad).

After a washing step with a Tris buffer 50 mM (pH8), M NaCl 0.5, proteinESM-1 was eluted with a solution of 3M MgCl₂, concentrated and dialysedagainst the same buffer on an ultrafree 30 device (millipore).

The eluted material was then quantified by immunodetection withanti-ESM-1 antibodies, and checked on SDS-PAGE using a coloration withcoomassie blue or alcian blue.

The purification of protein ESM-1 from human plasma was performedaccording to the following protocol.

800 ml of plasma supplied by the blood transfusion agency (Lille,France) were precipitated with a 60% ammonium sulfate solution anddialysed against a Tris buffer 50 mM (pH 8), 0.5 M NaCl. Theprecipitated and dialysed plasma extract was then passed over a 50 mlpre-column of the Affigel type (Biorad) before a passage over ananti-ESM-1 immunoaffinity column. The protein ESM-1 fixed on theimmunoaffinity column was recovered as described below.

The non-glycosylated form of ESM-1 (ESM/S137A) was purified in a singlestep by chromatography and immunoaffinity. The degree of purity ofglycosylated protein ESM-1 (ESM/WT) and of the non-glycosylated proteinreplaced on serine 137 (ESM/S137A) was checked by FPLC. The purifiedmaterial was free from endotoxins, as proved by the results of a limulusamebocyte lysate test (BIOwhitaker).

A.5 Immunoprecipitation, Immunoblotting and Sequencing.

The size of the different forms of ESM-1 was determined byimmunoprecipitation and immunoblotting from cell culture supernatantsand cell lysates. The cells were lysed in a buffer containing 0.5% ofNP40, a cocktail of anti-proteases (Boehringer Mannheim, Germany) in PBSfor 30 minutes at 4° C. with agitation.

The lysates were then centrifuged at 10.000 g for 15 min in order toobtain the clarified cell lysates.

The culture supernatants were filtered over a filter having a porediameter of 0.45 mm.

1 μg of ESM-1 monoclonal antibody produced by the hybridoma line MEP19or 1 μg anti-ICAM-1 monoclonal antibody (clone 164B) was added to theclarified lysate or to the cell culture supernatant and incubatedovernight at 4° C. with agitation.

50 μl of an anti-mouse immunoglobulin conjugated with agarose beads(sigma) were added at 4° C. over 90 min, before centrifugation andwashing with a lysis buffer and washing in PBS.

The beads were resuspended in 20 and 40 μl of SDS-PAGE buffer for 5 min,centrifuged, and the supernatants were analysed.

The samples were subjected to electrophoresis on SDS-PAGE gel, thentransferred onto a nitrocellulose membrane using standard procedures.

After a blocking step, the membranes were incubated for one hour with 1μl of an ESM-1 monoclonal antibody produced by the hybridoma line MEP14,washed, then incubated for 1 hour with an anti-Fc mouse secondaryantibody conjugated with horseradish peroxidase (marketed by SIGMA).After several washings revelation was performed using the detection kitECL marketed by Amersham.

For amino acid sequence analysis, purified protein ESM-1 was subjectedto electrophoresis on SDS-PAGE gel, then electrotransferred onto apolyvinylidene difluoride membrane (PVDF) marketed by MILLIPORE, thencoloured using 0.1% coomassie blue. The protein band at 50 kD wasexcised from the membrane and the N-terminal sequence was determined byEDMAN degradation on a protein sequencer of type ABI 473A.

A.6 Digestion of the Peptide Part of ESM-1 by Proteinase K

In order to determine the size of the glycosaminoglycan, purifiedprotein ESM-1 was digested with proteinase K with an enzyme:ESM-1 ratioof 1:50 (w/w) in a Tris buffer 10 mM, pH8, in the presence or absence of0.1% SDS at 56° C. for 3 hours. A quantity of bovine serum albumin (BSA)10 times?? greater than that of the protein ESM-1 was digested byproteinase K in order to verify its complete degradation. The sampleswere analysed on a 12% SDS-PAGE gel, followed by coloration withcoomassie blue and alcian blue.

A.7 Digestion of ESM-1 by Chondroitinases ABC, B, AC and C

In order to analyse the nature of the substitution of theglycosaminoglycan, purified protein ESM-1 was digested with severalchondroitinases: chondroitinases ABC (0.5 units/mg in buffer 100 mMTrisHCl, pH 8. 30 mM sodium acetate, pH 5.2 at 37° C. for 45 min),chondroitinase B (200 units/mg in buffer 20 mM Tris-HCl, 50 mM NaCl, 4mM CaCl₂, 0.01% BSA, pH 7.5 at 25° C. for two hours), chondroitinase AC(one unit per ml in buffer 250 mM Tris HCl, 75 mM sodium acetate, pH 7.3at 37° C. for two hours), chondroitinases C (80-120 units/ml in buffer50 mM Tris HCl , pH 8 at 25° C. for 3 hours). The samples were analysedby immunoblotting.

A.8 Anti-coagulant Activity.

The control plasma poor in platelets (PPP) was prepared from blood inthe presence of the anticoagulant sodium citrate (30 mM), bycentrifugation at 2500 g for 15 min. All the reagents were marketed bySTAGO Diagnostica (France). Three parameters were evaluated, by addingprotein ESM-1, buffer or heparin to the platelet-poor plasma.

a) APTT (Activated Partial Thromboplastin Time): this parameter exploresthe intrinsic route of blood coagulation (FI, FII, FV, FVIII, FIX, FX,FXI, FXII). The deficit or inhibition of one of these factors increasesthe coagulation time of the mixture PPP reagent, cephalin, activator,CaCl₂.

b) TCT (Thrombin Clotting Time): this parameter is analysed on a mixtureof platelet-poor plasma (PPP) in the presence of thrombin. With astandard concentration of thrombin, the coagulation time of the plasmais constant. Defects in the formation of fibrin induce an increase inthe coagulation time.

c) anti-Xa activity: the anti-Xa activity of heparin or other inhibitorsacting on factor FXa is detected by a competitive test. The samplestudied (PPP+ESM-1, +buffer or +heparin) is mixed with factor Fxa and aspecific chromogenic substrate of factor Fxa. The final coloration isinversely proportional to the concentration of inhibitor.

A.9 Test of Thrombin Generation

This sensitive global test can detect defects in plasma or plateletsinducing a delay or a reduction in thrombin generation. A plasma rich inplatelets (PRP) was prepared from blood in the presence of sodiumcitrate by centrifugation at 150 g for 10 min. The thrombin generationtest was performed, for each of the subjects, in samples in the absenceof ESM-1, with non-fractionated calcium heparinate (0.5 Ul ofanti-Xa/ml) or with 0.2 mg/ml, 0.5 mg/ml and 1 mg/ml of ESM-1 (finalconcentration).

The protein ESM-1 was added 10 min before the test.

At 37° C., 1 ml of plasma was mixed with 1 ml of CaCl₂ and a chronometerwas started. Aliquot fractions of 0.1 ml were taken from the reactionmixture each minute for 1 min.

The clot formed in the reaction mixture was regularly removed. Thealiquot fractions were mixed with 0.2 ml of fibrinogen (Sigma, 4/1000 inan Owren buffer at 37° C. and the coagulation time was measured for eachof the aliquot fractions).

The thrombin formed in the reaction mixture acts of the forminogens,inducing the formation of fibrin. The coagulation activity was maximalbetween 4 and 8 min then reduced due to the neutralization of thethrombin by the anti-thrombin.

A.10 Analytical Chromatography by Gel Filtration

50 μg of purified glycosylated ESM-1 (ESM/WT) and of purified nonglycosylated ESM-1 (ESM/S137A) in 50 mM Tris buffer, pH 8.5, 0.5 M NaClwere separated by liquid chromatography on a Superdex 200 column (forESM/WT) or Superdex 75 (for ESM/S137A) marketed by Pharmacia, using thechromatography system Biorad Biologic Chromatography System with a flowof 1 ml/min.

As standard, the following calibration kit of high and low molecularweight (Pharmacia Biotech) was used, ribonuclease A (bovine pancreas,13.7 kD), ovalbumin (43 kD) albumin (bovine serum, 67 kd), aldolase(rabbit muscle, 158 kD), ferrtine (horse spleen, 440 kD), thiroglobulin(bovine thyroid, 669 kD).

The molecular weight standards were separated using a buffer identicalto that used for the proteins ESM-1 and the separation was performedimmediately after the separation of the proteins ESM/WT and ESM/S137A.The elution time of the standard proteins was used to draw a standardlinear curve, Kav=f(log MR) in order to determine the apparent molecularweights of the proteins ESM/WT and ESM/S137A respectively.

Fractions of 1 ml were collected an protein ESM-1 was detected using aspecific immunoenzymatic test (ELISA).

B. Results

B.1 Post-translational Modifications of the Secreted Form of ProteinESM-1 Produced by Endothelial Cells and by Established Cell Lines.

In order to determine whether ESM-1 was matured as a secreted molecule,as suggested by the presence of an N-terminal amino acid sequencepredicted as a signal peptide, protein ESM-1 was purified from the cellline 293-ESM.

The N-terminal sequence of the 50 kD form indicated that the signalpeptide of 19 amino acids was cleaved at the predicted site, resultingin a mature polypeptide of ESM-1 of 165 amino acids beginning at thetryptophan residue in position 20 of sequence SEQ ID N^(o)1, theN-terminal sequence being WSNNYAVD-P.

ESM-1 was immunoprecipitated from culture supernatants of HUVEC, SV1,293-ESM and CHO-ESM cells, the analysed by immunoblotting.

It had previously been shown that in HUVEC cells supernatants, ESM-1migrated in the form of a diffuse band at around 50 kD.

A band similar in size was observed with the supernatants of SV1,293-ESM and CHO-ESM cells (FIG. 1A).

The molecular weight found was larger than the predicted molecularweight. This result suggested that the secreted form of ESM-1 hadundergone post-translational modifications. The fact that purifiedprotein ESM-1 was better coloured on SDS-PAGE gel with alcian blue thanwith coomassie blue suggested that ESM-1 was glycosylated (FIGS. 1B, 1C)rather than oligomerized across the disulfide bridge, because reductiveconditions did not modify the apparent molecular weight of ESM-1.

B.2. The Serine Residue in Position 137 (SEQ ID N^(o)1) is the Site ofO-glycosylation of ESM-1.

A computer analysis of the potential glycosylation sites identifiedthree putative sites of Olycosylation, respectively on the serine inposition 16, on the threonine in position 120 and on the serine inposition 127, but no site of N-glycosylation.

The threonine residue in position 120 and the serine residue in position137 were replaced by an alanine residue.

These mutants were transitorily expressed in the 293 cells.

Protein ESM-1 was then immunoprecipitated from cell lysates and culturesupernatants, and analysed by immunoblotting.

Protein ESM/T120A migrated at 50 kD, at a position similar to theapparent molecular weight of the wild form of ESM-1 (ESM/WT), as shownon FIG. 2A.

In contrast, protein ESM/S137A migrated at 22 kD corresponding to theintracellular form of ESM-1 (FIG. 2A), a molecular weight compatiblewith the predicted molecular weight of ESM-1.

The immunoprecipitations performed from transitorily transfected COS andCHO cells gave the same results, showing that only the serine residue inposition 137 constituted a site of glycoconjugation in all the cellmodels studied.

In order to determine the length of the glycosaminoglycan (GAG) ofESM-1, the peptide part of ESM-1 was completely digested by proteinaseK.

The treatment by proteinase K caused a change in the molecular weightfrom 50 kD to 25-30 kD (FIG. 2B). These results show that the band ofapparent molecular weight at 50 kD is compatible with the presence of apolypeptide of 22 kD which is glycoconjugated on the serine in position137 by a GAG chain of a mean size of 25-30 kD.

B.3 The GAG Chain of ESM-1 is Sensitive to Chondroitinase ABC

In order to characterize the GAG chain of ESM-1, protein ESM-1 was firstdigested by chondroitinase ABC. The treatment by chondroitinase ABCreduced the molecular weight of secreted protein ESM-1 to 22 kD (FIG.3A), suggesting that the carbohydrate of ESM-1 is a chain of thechondroitin type.

The profile is similar with protein ESM-1 purified from 293-ESM cellsand from the human endothelial cell line SVI. Because protein ESM-1circulates in the blood, we also studied the behaviour of protein ESM-1purified, from human plasma. The results showed a single principal bandof 50 kD, which had a molecular weight of 22 kD after treatment withchondroitinase ABC, as for all the other cell lines studied (FIG. 3A).Thus protein ESM-1 is a soluble proteoglycan containing a singlechondroitin sulfate chain.

B.4 The GAG Chain of ESM-1 is a Heterogeneous Chondroitin/dermatanSulfate Chain.

In order better to determine the type of saccharidic unit whichconstituted the GAG chain of ESM-1, several specific enzymes were used,such as chondroitinases B, AC and C.

The treatment with chondroitinase B reduced the apparent molecularweight from 50 kD to 22 kD (FIG. 3B).

A similar profile was observed after treatment of ESM-1 bychondroitinases AC and C (FIGS. 3C, D).

These different enzymatic treatments showed that the GAG chain of ESM-1contained different component units including a type of amino sugar,N-acetylgalactosamine, coupled to a differently sulfated iduronic orglucuronic acid.

These different units alternated in the chain, and were present at thebeginning of the chain, close to the N-terminal disulfated dissacharideswhich persisted in the protein part after digestion by thechondroitinase, because all the treatments with chondroitinase lead tothe same reduced apparent molecular weights of 22 kD.

B.5 Biological Activity of the Soluble ESM-1 Proteoglycan on Coagulation

Because protein ESM-1 is a secreted as a proteoglycan of thechondroitin/dermatan sulfate type by endothelial cells, and the dermatansulfate shows effects on thrombin generation in vitro DELORME et al.,(1996) and on coagulation, the anticoagulant potential of ESM-1 wasverified using the parameters APTT, TCT, anti-Xa activity and onthrombin generation.

The results are given in table 1 below.

TABLE 1 Biological activity of the ESM-1 proteoglycan on coagulationActivity APTT (dry) TCT (dry) Anti-Xa (UI/ml) PPP + buffer 30.6 16.5 0PPP + ESM-1 (0.2 μg/ml) 30.8 17.5 0 PPP + ESM-1 (0.5 μg/ml) 31 18.8 0PPP + ESM-1 (1 μg/ml) 31.8 20.7 0 PPP + heparin 89 39 0.45

The results in table 1 show that protein ESM-1 at different significantdoses from 0.2 mg/ml to 1 mg/ml did not change the different parameterstested.

The parameters APTT, TCT and anti-Xa activity were similar for plasmapoor in platelets (PPP) with the buffer or with protein ESM-1.

In the positive controls, the APTT, TCT and anti-Xa activities werehigher for PPP in the presence of heparin.

In addition, protein ESM-1 did not have an inhibitor effect on thethrombin generation test; no difference was observed according toconcentrations of 0.2 mg/ml, 0.5 mg/ml and 1 mg/ml of ESM-1 compared tothe control buffer, while heparin induced a delay in the formation ofthrombin (FIG. 4).

Example 2 Effect of Protein ESM-1 onto the Mitogenic Activity of FactorHGF/SF

A. Materials and Methods

The activity of stimulation of proliferation was determined by measuringthe incorporation of ³H thymidine by 293 cells.

The 293 cells were sown at a concentration of 1×10⁴ cells per well in96-well microplates of type TPP and maintained for 24 hours in DMEMculture medium supplemented with transferin and insulin.

The human recombinant HGF/SF was diluted in PBS containing 0.1% bovineserum albumin and added in water to 3 identical wells in order to obtaina final concentration of 50 ng/ml.

The recombinant proteins ESM/WT, ESM/S137A, the purified GAG chainderived from ESM-1 and decorin were added alone or in combination withfactor HGF/SF at doses of from 1 ng/ml to 2.5 μg/ml, simultaneously withthe addition of HGF/SF.

After 96 hours of culture, the cells were incubated with 0.5 μCi of ³Hthymidine per well for 16 hours and incorporation of ³H thymidine wasdetermined using a scintillation counter of the type Topcount MicroplateScintillation Counter (Packard).

The tests were performed on batches of three identical wells.

The cell viability was measured using the MTT reduction test.

B. Results

The effect of protein ESM-1 on the activity of factor HGF/SF wasstudied.

The incorporation of ³H-thymidine by. 293 cells was measured in thepresence of HGF/SF at 50 ng/ml alone or in combination with differentquantities of ESM/WT.

In a first batch of experiments, it was observed that HGF/SF alone at 50ng/ml induced a proliferation of the 293 cells at a level equal to about45% of the proliferation induced by the serum, while protein ESM/WTalone did not stimulate the proliferation of 293 cells.

In contrast, when it was combined with factor HGF/SF, protein ESM/WTconsiderably increased the proliferation of 293 cells induced by HGF/SFwith an increase of 162.3%, when the protein was tested at aconcentration of 2.5 μg/ml (FIG. 5).

This increase effect of protein ESM-1 on HGF/SF activity was dependenton the dose of ESM-1 and began to be significant at a dose of 10 ng/ml(FIG. 6).

In addition, the effect of protein ESM/WT was compared to the effect ofdecorin, another proteoglycan of the type chondroitin sulfate/dermatansulfate, on the mitogenic activity of factor HGF/SF. In contrast toprotein ESM/WT, decorin showed no activity of increasing theproliferation of 293 cells induced by factor HGF/SF (FIGS. 5, 6).

These results showed that protein ESM-1 had a specific effect on themitogenic activity of factor HGF/SF.

In order to examine the respective involvement of the protein part ofESM-1 and of the GAG chain on the activity of increasing the mitogeniceffect, the incorporation of ³H-thymidine by 293 cells in the presenceof HGF/SF supplemented with different concentrations of non-glycosylatedESM/S137A and of the GAG chain derived from of ESM-1 was measured.

The non-glycosylated form of ESM-1 was incapable of inducing aproliferation of the 293 cells, either in the presence or absence offactor HGFSF (FIG. 5), even when it was used at high concentration.

In contrast, the GAG chain purified from ESM-1 considerably increasedthe proliferation of 293 cells induced by factor HGF/SF, with a factorof increase close to 96.6%, compared to factor HGF/SF alone (FIG. 5).The pro-mitogenic effect of the GAG chain was less than that observedwith the wild form of protein ESM-1, but this effect was neverthelessdependent on the dose of GAG chain added (FIG. 6).

The results given above clearly show that protein ESM/WT increases theproliferation of 293 cells induced by factor HGF/SF and that thispro-mitogenic activity is specific and due to the GAG chain of thechondroitin sulfate/dermatan sulfate type of ESM-1.

In general, factor HGF/SF is expressed during the critical early periodsof human organogenesis from 6 to 13 weeks of gestation. The organs whichexpress the HGF/SF gene are particularly the liver, metanephric kidney,intestine and lung, each of these organs developing by inductiveinteraction between the mesenchyma and the epithelium. In addition,factor HGF/SF is an important factor in human renal multcystic dysplasia(TAKAYAMA et al., 1997) and in the appearance of malformation andhyperproliferation in the tubules. The results presented above show thatprotein ESM-1 significantly increases the proliferation of the cells ofthe embryonic kidney in the presence of HGF/SF while the nonglycosylatedform of protein ESM-1 has no effect. In addition, the GAG chain isolatedfrom protein ESM-1 is able to mimic the effects of the glycosylatedprotein ESM/WT. These results clearly show that the biological activityof ESM-1 on the function of factor HGF/SF is principally mediated by itsGAG chain. It may be noted that decorin, another proteoglycan of thechondroitin sulfate/dermatan sulfate type secreted by endothelial cellsand which is able to fix onto factor HGF/SF (CELLA et al., 1992) has noeffect on the activity of HGFSF. These comparisons show a specificity ofaction of protein ESM-1 on the activity of factor HGF/SF requiring acomposition of the GAG chain different from the GAG chain of theproteoglycans belonging to the family of proteoglycans with smallleucine-rich repeats.

In the kidney, protein ESM-1 is selectively detected in the distaltubules, a result which may be associated with the observation of apreferential localization of factor HGF/SF in the same part of thenephron in situations of human renal multicystic dysplasia (WEIDNER etal., 1993). These results indicate an application of protein ESM-1 inpathological disorders depending on factor HGF/SF, which has also beenshown as being associated with the development of cancers of the breast(RAHIMI et al., 1998), kidney (NATALI and al, (1996)) and lung (OTSUKAet al., 1998) and also in malignant melanomas (SIEGFRIED et al., 1998).Thus, factor HGF/SF is likely to favorize the extension of hyperplasiaand to generate cells with an invasive phenotype. Protein ESM-1 islikely to be involved in these phenomena of deregulated mitogenicactivities of factor HGF/SF.

Example 3 Preparation of an Antagonist Compound of Protein ESM-1 of theAntibody Type

In order to obtain anti-ESM-1 monoclonal antibodies directed against theN-terminal region of protein ESM-1 rich in cysteine residues, the nativeform of protein ESM-1 produced by the CHO cell line transfected by anexpression vector containing a DNA insert coding for protein ESM-1 waspurified.

The cDNA of ESM-1 was inserted into the eukaryotic expression vectorpcDNA3 (In vitrogen) then transfected in CHO cells with lipofectamine(GIBGO) according to the manufacturer's recommendation. 48 Hours afterthe transfection the cells were transplanted in the presence of aselection agent (G418, Gibco) at a dose of 1000 microgram/ml). After twoweeks of selection, the CHO cells resistant to G418 were cloned bylimiting dilution. The clones expressing ESM-1 were then selected andnamed CHO-ESM (deposited at the CNCM).

For the production, the CHO-ESM cells were cultured in suspension in amedium without foetal calf serum (medium CHO SFM II, Gibco). Thesupernatant was adjusted to pH 8 and passed over a DEAE-sepharose column(Pharmacia). The column was washed with a buffer 50 mM Tris, pH 8, 0.2 MNaCl. The ESM-1 molecule was eluted in a buffer 50 mM Tris, pH 8, 1 MNaCl. The eluate was then diluted 1:4 in a buffer 50 mM Tris, pH 8 andincubated in the presence of anti-ESM-1 monoclonal antibody (MEC4)immobilized on agarose (Biorad). After incubation overnight at 4° C.with agitation the agarose beads were washed with buffer 50 mM Tris, pH8, 0.2 M NaCl. ESM-1 was eluted with 3 M MgCl₂. The eluate wasconcentrated and dialysed in buffer 50 mM Tris, pH 8, 0.5 M NaCl andstored at −70° C.

Balb/C mice were immunized by injection of 10 μm of purified recombinantprotein ESM-1 per mouse, according to a standard immunization protocolin the presence of Freund's adjuvant.

The hybridoma cells secreting the anti-ESM-1 monoclonal antibodies wereobtained by fusion, screening and sub-cloning according to the techniquedescribed by BECHARD et al. (2000).

Five hybridoma cell clones were obtained and generically designated MEC(Mouse Monoclonal Antibody to ESM-1 produced by CHO Cells).

Four of the hybridomas selected were of isotypes IgG1, k, respectivelythe hybridomas designated MEC4, MEC5, MEC15 and MEC36.

One of the hybridomas was of isotype IgM,k, the hybridoma MEC11.

The hybrdoma cell clones were cultured in culture medium in the absenceof serum and the anti-ESM-1 antibodies were purified by chromatographyon a column of protein G-Sepharose marketed by Pharmacia (UPPSALA,Sweden).

Example 4 Preparation of an Antagonist Compound of Protein ESM-1 of thePolypeptide Type

The directed mutagenesis was performed with the kit marketed bySTRATAGENE under the reference Site-directed quick mutagenesis kit, usedaccording to the recommendations of the manufacturer.

Briefly, a pair of forward and reverse primers of strictly complementarysequences were synthesized, these primers comprising the nucleotidescoding for the mutated amino acid(s), or the complementary nucleotides,these nucleotides being localized in the centre of the sequence of theprimers which also comprise about 10 to 15 consecutive nucleotidescomplementary to the sequence to be amplified both on the 5′ and the 3′side of the central nucleotides.

After amplification by PCR, the amplified polynucleotides coding for themutant protein ESM-1 were inserted into the vector pcDNA3.

The following pairs of primers respectively were used:

a) For protein ESM-1 F115A

Forward primer: 5′-GCC TGA AAT TCC CCG CCT TCC AAT ATT CAG-3′. (SEQ IDN^(o) 3) Reverse primer: 5′-CTG AAT ATT GGA AGG CGG GGA ATT TCA GGC-3′.(SEQ ID N^(o) 4)

b) For protein ESM-1 F116A

Forward primer: 5′-CCT GAA ATT CCC CTT CGC CCA ATA TTC AGT AAC C-3′.(SEQ ID N^(o) 5) Reverse primer: 5′-GGT TAC TGA ATA TTG CGC GAA GGG GAATTT CAGT G-3′. (SEQ ID N^(o) 6)

c) For protein ESM-1 F115 F116A

Forward primer: 5′- CCT GAA ATT CCC CGC CGC CCA ATA TTC AGT AAC C-3′.(SEQ ID N^(o) 7) Reverse primer: 5′- GGT TAC TGA ATA TTG GGC GGC GGG GAATTT CAG G-3′-. (SEQ ID N^(o) 8)

Example 5 Pro-tumorigenic Activity of Glycosylated Protein ESM-1.

A. Materials and Methods

A.1. Cell lines : HEK T. HEK ESM/WT, HEK ESM/S137A, HEK ESM/69, HEKESM/71, HEK ESM/73.

The cell line HEK ESM/WT transfected stably with the cDNA coding for thewild form of ESM-1 (ESM/WT) was used. Four other cell lines wereobtained by transfection with the cDNA coding for the purified forms ofESM-1 obtained by directed mutagenesis of the wild type. The first ofthese, named HEK ESM/S137A, expressed the mutant non-glycosylatedprotein ESM-1, where an alanine has replaced serine 137, the major siteof O-glycosylation. The three other lines expressed a glycosylated formof ESM-1 whose protein part has been mutated. They were lines HEKESM/F115A (replacement of the phenylalanine in position 134, HEK ESM/71(replacement of the phenylalanine in position 135) and HEK ESM/F115A,F116A (double deletion/replacement 134-135).

Thus, six cell line producing different forms of ESM-1 were used:

-   -   control HEK, not secreting ESM-1;    -   Wild form of ESM-1: HEK ESM/WT;    -   Deglycosylated form of ESM-1: HEK ESM/S137A;    -   Glycosylated forms whose protein part has been mutated in the        region 115-116; HEK-ESM/69, HEK-ESM/71, HEK ESM/73.        A2. Murine Model of Xenogenic Tumours

The mice used were of type SCID (Severe Combined IMMUNO Deficiency).They were more precisely mice C.B.17 Scid/scid supplied by the animalservice of the Institut Pasteur of Lille. These mice had a recessiveautosomal mutation in their recombination system (Blunt., 1995). Thismutation causes the production of non-functional immunoglobulins and Tcell receptors (TcR) and B 5BcR) As a result, they do not possessfunctional T and B lymphocytes; these mice therefore tolerate non-selfand represent a model of choice for the development of xenogenictumours. The SCID mice used were young male mice aged from 3 to 5 weeks.For each of them, an intra-peritoneal injection of anti-ascialo GM-1antibodies (100 μg per mouse diluted in 200 μl of RPMI) was performed 24hours before injection with the different cell lines. These were rabbitpolyclonal antibodies (Wako Pure Chemical Industries, Ltd) directedspecifically against the asialo GM-1 antigen expressed by NK cells.Previous work has shown that the use of these antibodies in mudne modelsneutralizes the cytotoxic effect of the NK cells and encourages thetumoral grafting (Mather G et al. (1994).

Four batches of mice (10 to 15 mice per group) anaesthetized with ether,were then injected subcutaneously in the back. Each mouse received 1million cells diluted in 200 μl of RPMI. The injection of these cellsdefined the first day of the experiment (D0). For each mouse,macroscopic inspection of the point of injection in order to observe theappearance of a possible tumour, as well as measurement of body weight,was performed weekly. A blood sample (about 500 μl per mouse) was weeklytaken from the 5th week onwards, in order to determine the serum levelsof ESM-1 by an ELISA test (BECHARD D) et al., 2000). Ananatomo-pathological examination was performed on each mouse.

B. Results

B.1. Induction of Tumours in Mice by Glycosylated Protein ESM-1.

HEK cells were transfected with a vector possessing an insert containingthe cDNA coding for glycosylated wild protein ESM-1, designated ESM/WT.The HEK cells were injected subcutaneously into SCID mice aged 5 weeks.Each mouse had previously received an intraperitoneal injection ofanti-asialo GN-1 antibodies.

The percentage of tumours having a volume greater than 1 cm³ observed inthe mice at the eighth week following the injection of the transfectedHEK cells was analysed.

The results are given on FIG. 7.

On FIG. 7A, it can be observed that the injection of control HEK cellsdid not induce the appearance of tumours in mice In contrast, the HEKcells transfected with a DNA coding for glycosylated protein ESM-1induced numerous macroscopically visible tumours, of which about 95% hada tumoral volume greater than 1 cm³.

FIG. 7B illustrates the kinetics of appearance of tumours in mice whichhad received transfected HEK cells transfected with a DNA coding forglycosylated protein ESM-1. It can be observed that the mean tumoralvolume, expressed in cm³, increased continuously from the fourth weekfollowing the injection of the transfected HEK cells.

The experimental results presented in FIG. 7 clearly show thatglycosylated protein ESM-1 has a pro-tumoral activity.

The serum levels of protein ESM-1 were also measured in mice havingreceived control HEK cells and mice having received HEK cellstransfected with cDNA coding for protein ESM-1.

The results are shown on FIG. 8.

The results illustrated in FIG. 8A show that protein ESM-1 was not foundin the serums of mice having received control HEK cells. In contrast, aserum level of 40 to 50 nanograms per ml was found in mice havingreceived HEK cells transfected with cDNA coding for protein ESM-1 at theeighth week following injection of the cells.

The kinetics of the serum levels of ESM-1 in mice having receivedtransfected HEK cells expressing glycosylated protein ESM-1 (ESM/IT)were also analysed.

The results are given in FIG. 8B.

It can be observed that a detectable quantity of protein ESM-1 was foundin the serum of mice from the fifth week following the injection ofcells and that the serum level increased rapidly and continuously fromthe fifth to the twelfth week following injection of the cells.

The experimental results illustrated in FIG. 8 show that the tumourswhich developed in mice having received transfected HEK cells produceprotein ESM-1. In addition, the quantity of protein ESM-1 produced inthe circulation follows the kinetics of development of the tumours inthe mice.

Example 6 Pro-tumorigenic Activity of Different Forms of Protein ESM-1

A. Materials and Methods

The materials and methods used in this example are identical to thosedescribed for example 5.

B. Results

The HEK cells were transfected by vectors possessing a DNA insert codingrespectively for the nonglycosylated wild form of ESM-1 (ESM/WT), anon-glycosylated form of ESM-1 (ESM/S137A) and a glycosylated form ofESM-1 mutated at the phenylalanine residues in positions 134 and 135which have both been replaced by an alanine residue (ESM/73). Thedifferent transfected cells were injected subcutaneously into SCID miceaged 5 weeks and having previously received anti-asialo GM-1 antibodies.

The percentage of tumours macroscopically visible having a tumoralvolume greater than 1 am³ in the different batches of mice was analysed.The results are shown on FIG. 9A.

The results of FIG. 9A show that only the glycosytated protein ESM-1 isable to induce tumours in mice. Neither the non-glycosylated ESM-1 northe glycosylated ESM-1 mutated at the phenylalanine residues inpositions 134 and 135 induced the development of tumours in SCID mice.

The serum levels of protein ESM-1 circulating in the different batchesof mice were also measured. The results are given in FIG. 9B.

The results on FIG. 9B show that detectable levels of serum proteinESM-1 could be measured, at the eighth week following injection of thecells, only in the mice having received the HEK cells expressingglycosylated protein ESM-1 (ESM/WT).

Neither the mice injected with cells expressing the non-glycosylatedprotein ESM-1 (ESM/S137A), nor the mice having received the glycosylatedand mutated protein ESM-1 HEK-ESM/F115A, F116A) produced protein ESM-1.

The overall results presented in this example confirm thepro-tumorigenic activity of glycosylated protein ESM-1.

The results also show that the non-glycosylated forms of protein ESM-1or the mutant forms of protein ESM-1 can behave as antagonists of thisprotein and possess preventive and/or curative power with regard tocancerous pathologies.

Example 7 Determination of Circulating Protein ESM-1 in PatientsSuffering from Broncho-pulmonary Cancers at Different Stages ofDevelopment

A. Materials and Method

The immunodetection test consisted of an immuno-enzymatic test of the“sandwich” type whose general characteristics are identical to thosedescribed by BECHARD et al. (2000).

The anti-ESM-1 monoclonal antibody produced by the hybridoma lines MEP14(CNCM N^(o)I-1942) was diluted to a concentration of 5 μg/ml in acarbonate buffer 0.1 M, pH 95, and adsorbed overnight at +4° C. on a96-well plate (plate E.I.A./R.I.A., Costar, Cambridge, Mass., USA).

The plate was saturated for one hour at laboratory temperature with avolume of 200 μl/well of PBS buffer containing 0.1% of bovine serumalbumin and 5 mM of EDTA, then washed twice with an ELISA buffer (thePBS buffer above supplemented with 0.1% Tween 20).

A calibration was performed with protein ESM-1 purified according to thetechnique described by BECHARD et al. (2000).

The blood samples were serially diluted (1:2 to 1:128), in an ELISAbuffer and incubated on an ELISA plate for one hour at laboratorytemperature.

The wells were washed three times with an ELISA buffer, then incubatedfor 1 hour at laboratory temperature with a second monoclonal antibodydirected against ESM-1, the antibody MEC15 (CNCM N^(o)I-2572) at aconcentration of 0.1 μg/ml in 100 μl of buffer per well.

After three washings, a biotinylated rat monoclonal antibody ratdirected against mouse IgG1 (marketed by PHARMINGEN) diluted in an ELISAbuffer was added and left to incubate this second antibody for one hour.

After three washings in the ELISA buffer, the wells were incubated witha streptavidine-peroxidase conjugate at a dilution 1:10.000 v/v(marketed by ZYMED).

After 30 minutes of incubation-with the streptavidine-peroxidaseconjugate, three washings of each well were performed with an ELISAbuffer, then two washings in a PBS buffer.

The streptavidine-peroxidase conjugate was revealed with the substrateTMB marketed by SIGMA (Saint-Louis, Mo., USA) in the presence of 255 μlof H₂O₂ for 30′.

The revelation reaction was stopped by addition of a volume of 100 μl ofH₂SO₄ 2N.

The plate was read using a spectrophotometer (anthos labtec LP40.France) at a wavelength of 405 nanometres.

The plasma or serum concentration of protein ESM-1 was calculated fromthe optical density measurements and expressed in nanograms per ml.

B. Results

The concentration of protein ESM-1 circulating in the serum of differentpatients -with broncho-pulmonary cancer at different stages development,respectively at stage I, II, IIIA, IIIB and IV according to theinternational classification TNM defined below:

T=size of the tumour (T1:<1 cm; T2: between 1 and 3 cm; T3:>3 cm.

N=ganglion nodule (NO if not invaded; N1 if invaded).

M=metastasis at distance (MO if no metastasis; M if metastasis).

The patients suffering from cancer at stage I had a serum concentrationof protein ESM-1 of 1.43+/−0.76 nanograms/ml (n=3).

The patients suffering from a bronchopulmonary cancer at stage If had aserum concentration of protein ESM-1 of 0.72+/−0.39 nanograms/ml (n=3).

The patients suffering from a bronchopulmonary cancer at stage IIIA hada concentration of circulating protein ESM-1 of 0.9+/−0.53 nanograms/ml(n=2).

The patients suffering from a bronchopulmonary cancer at stage IIIB hada concentration of circulating protein ESM-1 of 3.1+/−2.17 nanograms/ml(n=3).

The patients suffering from a bronchopulmonary cancer at stage IV had aconcentration of circulating protein ESM-1 of 3.1+/−1.91 nanograms/ml(n=11).

The results given above show that the serum levels of protein ESM-1increase as a function of the stage of development of the cancer. Aclear relation is thus demonstrated between the level of production ofprotein ESM-1 in the blood circulation and the severity of a cancer in apatient.

Example 8 Anti-tumoral Activity of an Antagonist Compound of ESM-1 ofthe Antibody Type

A. Materials and Method

MEP08 monoclonal antibodies were injected intraperitoneally at a dose of400 μg from the second following the inoculation of HEK/ESM-WT cells.The injections were repeated weekly for 12 weeks. A control antibody,MEP-14, was used under the same conditions. The mice were sacrificedwhen their tumoral volume was greater than 6 cm³. (n>8 mice in eachgroup). The figure shows the percentage of surviving mice in each of thegroups.

B. Results

To the extent that the phenylalanine in position 115 is necessary fortumoral development, it comprises a new therapeutic target. For thisreason the anti-ESM-1 monoclonal antibodies MEP-08, produced by thehybridoma line MEP-08 deposited at the CNCM under the n^(o)I-1941,directed specifically against this region, were produced and injectedinto the group of HEK-ESM/WT mice. The object was to study the role ofthe peptide of ESM-1 in the tumoral development and to evaluate apossible therapeutic effect. In order to eliminate an anti-tumoraleffect depending on the fragment Fc of the antibody (reaction of ADCC),a control antibody of the same isotype but recognizing a differentepitope was used in parallel and under the same conditions.

FIG. 10 shows that the early injections of MEP-08 antibodiessignificantly increased the survival of the mice by nearly 60% while theMEP-14 antibodies had no effect. These first results show that this is aspecific action linked to the fragment Fab of the antibody directedspecifically against the phenylalanine in position 115 and confirm theinvolvement of the peptide in the tumoral growth. It is surprising toobserve that this effect on the survival reduces when the antibodies areadministered later.

Whichever week the injections begin, the antibodies can delay or preventthe tumoral growth. This anti-tumoral effect remains more pronouncedwhen the antibodies are used earlier.

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1. A method of inhibiting or blocking the tumorigenic capacity of ESM-1 protein in a cancer patient, wherein said method comprises administering to the patient a pharmaceutical composition comprising (i) a monoclonal antibody produced by the hybridoma line deposited at the Collection National de Cultures de Microorganismes of the Institut Pasteur under accession No. I-1941, also named MEP08, in an amount that inhibits or blocks the tumorigenic capacity of ESM-1 protein in vivo, and (ii) one or more pharmaceutically acceptable vehicles. 